Nucleosides for imaging and treatment applications

ABSTRACT

Methods of diagnosing and/or of treating tumors by administering a nucleoside analogue which is activated by thymidylate synthase and/or thymidine kinase enzyme into a diagnostic or toxic metabolite, and uridine analogue compounds, and compositions of same having a pharmaceutically acceptable carrier. For diagnostic applications, compounds containing a label and methods of use of such compounds are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.09/530,276, filed Apr. 28, 2000; which is a national stage applicationof International Patent Application No. PCT/US98/23109, filed Oct. 30,1998; which claims the priority of U.S. Application No. 60/063,587,filed Oct. 30, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods, compounds, and compositionsfor diagnosing and/or treating tumor cells with anti-tumor agentsactivated by thymidylate synthase (TS) and/or thymidine kinase (TK). Inaddition, the present invention relates to the preparation and use ofpositron emitting nucleoside analogues for use in imaging applications.The nucleoside analogues used in imaging applications may be of the typeactivated by TS or, in other embodiments, may not require activation byTS. More particularly, the present invention relates to methods fordiagnosing and/or treating tumor cells by administration of compoundssuch as nucleoside analogue prodrugs and related compounds orcompositions containing these in an effective amount to identifysusceptible tumors in biopsy specimens or via external imaging, and thenproceeding to reduce or inhibit the replication or spread of tumorcells.

2. Technology Review

Thymidylate synthase (TS) is an essential enzyme for DNA synthesis. Itis, however, more abundant in tumor cells than in normal tissues. Fordecades, research and clinical studies have been directed towardsinhibition of TS in order to shrink tumors. In some instances, thisstrategy has been modestly successful, for example, fluorouracil andfloxuridine are utilized in the treatment of breast, colon, pancreas,stomach, ovarian, and head/neck carcinomas as disclosed by Chu E,Takimoto C H. “Antimetabolites.” In: DeVita V T Jr., Hellman S,Rosenberg S A, editors, Cancer: Principles and Practice of Oncology,Vol 1. 4th ed. Philadelphia: Lippincott, 1993:358-374.

Unfortunately, most tumors are inherently resistant to this strategy,and even those tumors, which are initially sensitive, develop resistanceduring the course of treatment as reported by Swain S M, Lippman M E,Egan E F, Drake J C, Steinberg S M, Allegra C J, in “Fluorouracil andHigh-Dose Leucovorin in Previously Treated Patients with MetastaticBreast Cancer,” J. Clin. Oncol, 1989; 7:890-9. Recent applications ofmolecular probes for TS have demonstrated a consistent relationshipbetween resistance and high expression of TS as noted in the followingarticles: Johnston P G, Mick R, Recant W, Behan K A, Dolan M E, Ratain MJ, et al. “Thymidylate Synthase Expression and Response to NeoadjuvantChemotherapy in Patients with Advanced Head and Neck Cancer”, J. Natl.Cancer Inst. 1997; 89:308-13; Lenz H J, Leichman C G, Danenberg K D,Danenberg P V, Groshen S, Cohen H, Laine L, Crookes P, Silberman H,Baranda J, Garcia Y, Li J, Leichman L, “Thymidylate Synthase mRNA Levelin Adenocarcinoma of the Stomach: A Predictor for Primary Tumor Responseand Overall Survival”, J. Clin. Oncol. 1996; 14:176-82; Johnston P G,Lenz H J, Leichman C G, Danenberg K D, Allegra C J, Danenberg P V,Leichman L, “Thymidylate Synthase Gene and Protein Expression Correlateand Are Associated with Response to 5-Fluorouracil in Human Colorectaland Gastric Tumors”, Cancer Res 1995; 55:1407-12; Leichman L, Lenz H J,Leichman C G, Groshen S. Danenberg K, Baranda J, et al, “Quantitation ofIntratumoral Thymidylate Synthase Expression Predicts for Resistance toProtracted Infusion of 5-Fluorouracil and Weekly Leucovorin inDisseminated Colorectal Cancers: Preliminary Report from an OngoingTrial”, Eur. J. Cancer 1995; 31A: 1305-10. Kornmann M, Link K H, StaibL., Danenberg P V., “Quantitation of Intratumoral Thymidylate SynthasePredicts Response and Resistance to Hepatic Artery Infusion withFluoropyrimidines in Patients with Colorectal Metastases”, Proc. AACR38:614, 1997.

A new generation of drugs designed to inhibit TS is reported byTouroutoglou N, Pazdur R. in “Thymidylate Synthase Inhibitors”, Clin.Cancer Res., 1996; 2:227-43, to be currently in final stages of clinicaltesting. Despite the enormous resources which are being expended toimprove the effectiveness of first-generation TS inhibitors, neither theexisting drugs nor this new set of compounds are effective in tumorswhich have a high level of TS activity. Presently, once a tumor hasbecome resistant due to -high levels of TS, there is no specific therapyavailable.

Instead of inhibiting TS, the present inventors hypothesized that is waspossible to use this enzyme to activate uridine analogue prodrugs intomore toxic thymidine analogues. The present inventors have previouslydemonstrated in Molecular Pharmacology, 46: 1204-1209, (1994) in anarticle entitled, “Toxicity, Metabolism, DNA Incorporation with Lack ofRepair, and Lactate Production for1-2′Fluoro-2′deoxy-β-D-arabinofuranosyl)-5-iodouracil (FIAU) in U-937and MOLT-4 Cells” that 1-(2′Fluoro-2′deoxy-β-D-arabinofuranosyl)-uracil(FAU) was phosphorylated intracellularly by intact U-937 and MOLT-4cells to FAU monophosphate (FAUMP), converted to its methylated form,5-methyl-FAUMP (FAUMP), and incorporated into DNA. These priorobservations suggested that FAU would be an appropriate prototype fortesting the cytotoxic potential of TS-activated prodrugs. It is to beunderstood that the former study produced data for different purposesand does not directly address the present discovery. To demonstrate thevalidity of the present concept, the inventors: (1) determined that TSis the enzyme which catalyzed the methylation; (2) examined the netformation rates of methylated species in a variety of cells; and (3)correlated the net formation rates of methylated species with cytotoxiceffects.

Among pyrimidine nucleosides, 2′-deoxyuridine (dUrd) analogues are lesstoxic than their corresponding thymidine (dThd) analogues as indicatedby Kong XB, Andreeff M, Fanucchi M P, Fox J J, Watanabe K A, Vidal P,Chou T C, in “Cell Differentiation Effects of2′-Fluoro-1-beta-D-arabinofuranosyl Pyrimidines in HL-60 Cells.” LeukRes, 1987; 11:1031-9. The present inventors theorized that followingentry into the cell and phosphorylation, an analogue of dUrd would serveas a selective prodrug if TS can methylate it to generate thecorresponding dThd analogue. Thus, tumors which are resistant to TSinhibitors, because of high levels of TS, would be particularlysensitive to these deoxyuridine (dUrd) analogues, because they would bemore efficient in producing the toxic thymidine (dThd) species. Thisstrategy is completely novel, since it is entirely different from allprior approaches towards TS as an antitumor target. Contrary to previousresearch and clinical studies which are directed towards the inhibitionof TS in order to shrink tumors, the present invention utilizes TS toactivate uridine analogue prodrugs into the more toxic thymidineanalogues to reduce or inhibit tumor cells, especially tumor cells whichare inherently resistant to or develop resistance to existing therapies.The present invention is additionally highly complementary to all priorapproaches towards inhibition of TS as an antitumor target.

Further, because success of therapy with drugs such as FAU or itsanalogues is related to extent of incorporation into DNA, the analysisof DNA can provide diagnostic information regarding the optimal therapyfor a tumor. Thus, by examining a biopsy specimen of tumor, or byexternally imaging tumors, it can be predicted whether therapy with FAUor related compounds would be successful, or whether alternate therapyshould be used.

In addition to assessing tumor therapy, there are a variety of othermedical circumstances in which it is important to determine theproliferation rate (growth) of cells within a particular tissue in thebody. These include: assessment of bone marrow function (e.g., aftertransplantation and/or stimulation with growth factors), regeneration ofthe liver following surgery or injury, and expression of enzyme functionfollowing gene therapy.

Traditional approaches to determine growth rate have been invasive;i.e., have required obtaining a biopsy from the patient. In addition tothe discomfort and risks associated with biopsy procedures, only a smallsample of tissue is obtained. Thus, biopsies carry the inherent risk ofmis-diagnosis as the small sample may not be representative of theentire region. Thus, there is a need in the art for other methodologiesto determine the growth rate of tissues.

Non-invasive, external imaging methods avoid the need for biopsies, andalso have the capability of scanning large areas of the body, indeed,the entire body if necessary. Since growth (proliferation) requires thesynthesis of DNA from nucleosides, administration of nucleosides whichhave been radiolabeled with a positron emitter provides the ability toexternally monitor events occurring within the body by use of imagingtechnologies such as a PET (Positron Emission Tomograph) scanner, orother photon-detecting devices such as SPECT (Single Photon EmissionComputed Tomograph), or gamma cameras.

These imaging technologies are only limited by the availability ofprobes whose biological fates provide information as to theproliferative state of the tissue examined. Thymidine is a particularlyuseful probe for monitoring growth/DNA synthesis, because it is the onlynucleoside for which direct incorporation of exogenously appliednucleoside into DNA is common by “salvage” pathways. There is nodependence upon the ribonucleotide pathways for the incorporation ofthymidine. Thymidine itself is unsuitable as a probe in these imagingtechnologies, since the molecule is rapidly degraded in the body.Analogues of thymidine such as FMAU and FIAU are excellent imagingprobes, because they: 1) completely follow thymidine pathways forincorporation into DNA; 2) are not degraded by catabolic enzymes; and 3)can be labeled with ¹⁸F, the most desirable atom for positron imaging.

Imaging probes incorporating other positron emitting moieties have beenused in the prior art. For example, a synthesis for ¹¹C-FMAU has beenreported. However, there are a number of practical limitations dictatedby the 20-minute half-life of ¹¹C. Probe molecules containing ¹¹C mustliterally be prepared on-site and used within an hour. This requirementmakes it unfeasible to have a regional preparation center and ship themolecules to surrounding medical facilities. Thus, every facilitydesiring to perform imaging studies using a ¹¹C-labeled probe must haveon site the cyclotron facilities to prepare the isotope. An additionallimitation of ¹¹C-containing labels arises when the biological phenomenarequires more than an hour for full expression. The short half life of¹¹C means that insufficient ¹¹C would remain to be imaged in thesesituations.

In addition to ¹¹C-containing probes, probes labeled with ¹⁸F are knownin the prior art. ¹⁸F-fluorodeoxyglucose (FDG), a currently employedimaging probe, is synthesized and distributed from a regional facilitymaking it more easily available for imaging purposes. Further,nucleoside analogues incorporating ¹⁸F in positions other than those ofthe present invention, for example ¹⁸F at the 5 position of uracil, havebeen reported.

Notwithstanding the existence of the probe molecules discussed above,there exists a need in the art for probe molecules for use in externalimaging technologies. In addition, a need remains in the art foradditional therapeutic modalities for the treatment of cellproliferation disorders. These and other needs have been met by thepresent invention.

SUMMARY OF THE INVENTION

The present invention provides compounds, compositions, and methods ofdiagnosing and/or treating tumors. The compounds of the presentinvention include nucleoside analogues which are activated bythymidylate synthase and/or thymidine kinase enzymes in an effectiveamount for diagnosis or to reduce or inhibit the replication or spreadof tumor cells. These compounds and compositions comprising thesecompounds are easily administered by different modes known in the artand can be given in dosages that are safe and provide tumor inhibitionat the relevant sites.

The present invention includes nucleoside analogues containing apositron emitting label moiety for use in imaging applications. Theseanalogues may be synthesized so as to require activation by TS prior toincorporation into DNA and subsequent imaging. In alternativeembodiments, the analogues of the present invention will not requireactivation by TS when used for imaging applications. In otherembodiments, the analogues may be used for imaging applications eventhough not incorporated into DNA.

Accordingly, it is the object of the present invention to providecompounds, compositions, and methods to identify susceptible tumors inbiopsy specimens or via external imaging, and/or inhibit or reduce thereplication or spread of tumor cells.

It is another object of the present invention to provide a treatment fortumors and other diseases characterized by abnormal cell proliferationby administrating these compounds or compositions either alone or incombination with other agents that inhibit tumor growth and/or withother classes of therapeutics used to treat such diseases.

It is another object of the present invention to assess the impact ofother treatments (e.g., by radiotherapy or other drugs) upon tumorgrowth. In preferred embodiments, the treatments will be drugs intendedto inhibit thymidylate synthase.

It is an object of the present invention to provide compounds andmethods useful for external imaging applications. In preferredembodiments, the invention includes the selection, preparation, and usesof nucleosides labeled with fluorine-18 (¹⁸F), a positron emitter. Themethods of the present invention permit treatment individualizationusing surrogate markers such as external imaging. Other embodiments ofthe invention may be useful in selecting the most effective drugs to beused against tumors in humans.

It is an object of the invention to provide a method that can beutilized to monitor and assess the efficacy of supportive treatments. Inpreferred embodiments the supportive treatments may be bone marrowtransplant and/or stimulation by growth factors. In other preferredembodiments, the present invention may be used to monitor and assess thecourse of liver regeneration after surgery or injury.

It is an object of the present invention to provide a method formonitoring the expression of genes introduced in gene therapyapplications.

Other features and advantages of the present invention will be apparentfrom the following description of preferred embodiments. These and otherobjects, features and advantages of the present invention will becomeapparent after a review of the following detailed description andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the general structure for many TS substrates. For theendogenous nucleoside, dUrd, W═X═Y═H, and Z═OH. FAU has only a singlesubstitution, W═F. A phosphate group attached to the sugar at the5′-position is also required. The corresponding thymidine analogues havea methyl group (—CH₃) at the 5-position of the uracil base.

FIG. 2 graphically depicts the effect on cell growth of 72-hr continuousexposure to: (A) FAU, or (B) FMAU. Cell designations:

CEM=; MOLT-4=◯; RAJI=▾; U-937=∇; K-562=▪; L1210=□.

FIG. 3 graphically depicts the association of DNA incorporation witheffect on cell growth. (A) FAU, (B) FMAU.

CEM=; MOLT-4=◯; RAJI=▾; U-937=Δ; K-562=▪; L1210=□

FIG. 4 graphically depicts the relative sensitivity of cell lines togrowth-inhibition by FAU compared with the activation potential for TS,measured independently as relative dehalogenation of IdUrd. The mostsensitive cell lines (U-937, CEM, MOLT-4) have 50% or moredehalogenation. The least sensitive lines (Raji, L1210) have 15% orlower.

CEM=; MOLT-4=◯; RAJI=▾; U-937=∇; K-562=▪; L1210=□

FIG. 5 graphically depicts FAUMP conversion to FMAUMP by TS in U-937cell extracts, as demonstrated by the accumulation of tritiated water.The rate of conversion to FMAUMP was about 1% of the rate of dTMPformation from dUMP. Similar results were obtained for the other celllines, with a range of 0.97-1.5%.

FIG. 6 graphically depicts effect on cell growth of 72-hr continuousexposure to: (A) ara-U, or (B) ara-T. Cell designations:

CEM=; MOLT-4=◯; RAJI=▾; U-937=∇; K-562=▪; L1210=□

FIG. 7 graphically depicts effect on cell growth of 72-hr continuousexposure to: (A) dUrd, or (B) dThd. Cell designations:

CEM=; MOLT-4=◯; RAJI=▾; U-937=∇; K-562=▪; L1210=□

DETAILED DESCRIPTION OF THE INVENTION

Tumor cells with high levels of thymidylate synthase (TS) represent acommon therapeutic challenge for which no treatment strategy iscurrently available. One aspect of the present invention is that thegrowth of tumor cells with high TS can be preferentially inhibited witha uridine and/or a deoxyuridine (dUrd) analogue. As used hereinafter,uridine analogue is seen to include uridine and deoxyridine andderivatives of both. Further, since tumors can vary widely, theidentification of tumor cells with high levels of TS provides diagnosticinformation to select appropriate therapy for individual tumors. UsingFAU as a prototype this concept has been successfully demonstrated.

The following SCHEME I illustrates the generalized structure for dUrdanalogues and their intracellular activation pathways. For theendogenous nucleoside, dUrd, W═H. FAU has the substitution, W═F. Aphosphate group is attached to the sugar at the 5′-position by thymidinekinase (TK) to form dUMP or its analogue, FAUMP. Subsequently, TSattaches a methyl group at the 5-position of the base to generatethymidylate, dTMP, or its analogue, FMAUMP.

The present inventors demonstrated that FAU was converted into FMAUnucleotides and incorporated as FMAU into cellular DNA. In particular,the monophosphate of FAU, FAUMP was converted by TS in cell extracts tothe corresponding dThd form, FMAUMP. Incubation of FAU with tumor celllines in culture inhibited their growth to a variable extent, dependingupon the efficiency of activation via TS. This is the firstdemonstration that cells with high levels of TS activity can be morevulnerable to therapy than cells with low TS activity.

Wide variation among cell lines was observed in growth inhibition, andalso relatively shallow slopes for the response versus extracellularconcentration curves (FIGS. 2A, 2B). As a consequence, extracellularconcentration of FMAU or especially FAU was a weak predictor ofcytotoxicity, In contrast, the variation among cell lines in IC50related to % replacement of dThd in DNA by FMAU was quite small (FIG.3). Further, there were steep response curves for growth inhibitionversus incorporation of drug (as FMAU) into DNA. Further, there wassimilarity among cell lines in toxicity at similar values for %replacement of dThd in DNA by FMAU. Thus, for equal exposure to theprodrug, selective toxicity could be related to differences in the rateof conversion to dThd analogues by TS. However, although conversion byTS is a necessary condition for toxicity, it is not sufficient.Opportunistic utilization of elevated TS activity also relies upon othersteps, especially kinases and polymerases, as well as competition withendogenous synthesis. Growth inhibition ultimately depends upon the netaction of all the pyrimidine pathways.

These data in FIG. 3 also demonstrate a use of deoxyuridine analoguesfor diagnostic applications. Tumors with high uptake of FAU andincorporation into DNA after methylation via thymidylate synthase couldbe imaged externally, e.g., by use of ¹⁸F-labeled FAU with positronemission tomography (PET). Alternatively, a dose of FAU could beadministered prior to a tumor biopsy, and incorporation into DNAdetermined with the same techniques used for the cell culture samples inFIG. 3. By either modality, tumors with high DNA incorporation would beexcellent candidates for therapy with FAU or related analogues, andtumors with low DNA incorporation should be treated with some othertherapy.

It is possible that FAU has autonomous biologic effects separate fromFMAU nucleotides. However, there were several indications that formationof FMAU by TS was sufficient to explain the majority of observedeffects. In the present invention, comparison to the direct use of FMAUdemonstrated that the toxic effects were dominated by FMAU nucleotides,especially similarity in DNA relationships. In addition, the relativesensitivity of cell lines to growth-inhibition by FAU was compared withthe activation potential for TS, measured independently as relativedehalogenation of IdUrd (FIG. 4). The most sensitive cell lines (U-937,CEM, MOLT-4) have 50% or more dehalogenation. The least sensitive lines(Raji, L1210) have 15% or lower dehalogenation.

Nonetheless, under other experimental conditions, if there aredifferences among cells in transport, phosphorylation, or relatedpathways, then these factors can also influence response in addition toTS activity. A major advantage of imaging tumors with labeled FAU isthat it detects the end-product of all these processes, which shouldtranslate into prognostic value.

Further, an alternative or additional approach to diagnosis is suggestedby interpretation of the data in FIG. 4. Prior to biopsy, a dose oflabeled IdUrd, either radiolabeled or more preferably labeled withstable isotopes, can be given to a patient. Dehalogenation can bedetermined from the DNA in the tumor biopsy and used to guide therapy.

The present invention provides a promising avenue of attack for commonhuman tumors which have previously been resistant to therapeuticapproaches. Although FAU was used to demonstrate the principle, FAU wasnot very potent, and may not necessarily be the optimal compound in itsclass. The rate of methylation by TS was rather low, only 1% comparedwith the endogenous substrate, dUMP. Despite this low rate, substantialamounts of FMAU were incorporated into DNA and toxicity was observed.

If FAU is not ideal, there are many other synthetic modifications ofdUrd which can also serve as TS substrates. For example, cell culturedata were obtained for uracil arabinoside (ara-U) and its methylatedanalogue, thymine arabinoside (ara-T). As shown in FIG. 6, the patternsof toxicity for ara-U and ara-T are very similar to those for FAU andFMAU in FIG. 2, suggesting a similar mechanism, i.e., methylation.Further, the endogenous compounds, deoxyuridine (dUrd) and thymidine(dThd) also display the same pattern of cell culture toxicity (FIG. 7).

Accordingly, the present invention includes compounds, compositions andmethod for diagnosis and treatment of tumors. One embodiment of thepresent invention is the use of uridine analogues or related compoundsas disclosed herein to inhibit tumor formation. The present inventionalso includes compounds which have anti-tumor activity. The presentinvention also comprises a method of treating tumor formation in humansor animals comprising the steps of administering to the human or animalhaving tumors, a composition comprising an effective amount of a uridineanalogue which is capable of inhibiting tumor growth.

It is common practice to treat tumors empirically without diagnosticinformation regarding the sensitivity of the specific tumor to aparticular drug. Thus, FAU or related compounds could be used directlyto treat tumors of a class known to have high levels of TS.Alternatively, therapy with FAU or related compounds could begin afterfailure of conventional TS inhibitors, with the inference that TS levelsare elevated. A preferred approach would use biopsy or external imaginginformation to guide therapy selection, by diagnosing which tumors wouldbe susceptible to FAU or related compounds, and which should usealternate approaches.

Tumor inhibiting and/or diagnosing compounds that can be used inaccordance with the present invention include those having the followinggeneral formula:

wherein:

A=N, C;

B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C₁-C₆), alkoxy (C₁-C₆);

D=O, S, NH2;

E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy,substituted alkoxy, halogen, or any substituent which is readily cleavedin the body to generate any one of the before listed groups;

G=substituted or unsubstituted cyclic sugar, substituted orunsubstituted acyclic sugar, substituted or unsubstituted mono, di, ortri-phospho-cyclic-sugar phosphate; substituted or unsubstituted mono,di, or tri-phospho-acyclic-sugar phosphate; substituted or unsubstitutedmono, di, or tri-phospho-cyclic sugar analogues, substituted orunsubstituted mono, di, or tri-phospho-acyclic sugar analogues whereinthe substituents are alkyl (C₁ to C₆), alkoxy (C₁ to C₆), halogen.

The present invention also features methods of inhibiting tumor growthin mammals by administering a compound according to the above formula ina dosage sufficient to inhibit tumor growth.

The preferred compounds are:

wherein:

A=N, C;

B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C₁-C₆), alkoxy (C₁-C₆);

D=O, S, NH2;

E=H, ∥ |

W, X, Y, Z=H, hydroxy, halogen, alkyl (C₁-C₆), alkoxy (C₁-C₆), a labelcontaining moiety or a label;

J=C, S; and

K=O, C.

In preferred embodiments for anti-tumor activity, E may be H,

In other preferred embodiments, W is a halogen. In a most preferredembodiment, W is Fluorine and E is H, methyl, iodine or a substituentreadily cleaved by the body to generate one of these groups. However,other embodiments are within the scope of the present invention.

It is to be understood that the compounds of the present invention canexist as enantiomers-and that the racemic mixture of enantiomers or theisolated enantiomers are all considered as within the scope of thepresent invention.

The compounds of the present invention can be provided aspharmaceutically acceptable compositions or formulations usingformulation methods known to those of ordinary skill in the art. Thesecompositions or formulations can be administered by standard routes. Ingeneral, when used to treat cell proliferative disorders, the dosage ofthe compounds will depend on the type of tumor, condition being treated,the particular compound being utilized, and other clinical factors suchas weight, condition of the human or animal, and the route ofadministration. It is to be understood that the present invention hasapplication for both human and veterinarian use.

Any of these compounds can also be used to provide diagnosticinformation regarding the tumors. For example, if a patient is having abiopsy of his/her tumor, a dose of FAU or related compound can beadministered, with or without the use of a radiolabeled atom, or morepreferably a stable isotope of the naturally occurring atom, and the DNAfrom the biopsies treated as in the cell culture experiments.

Any generally known and acceptable radioisotopes or stable isotope of anaturally occurring atom can be utilized in the present invention.However, ¹⁴C and ³H are preferred radioisotopes and stable isotopiclabels such as ¹³C, ²H, or ¹⁵N are most preferred.

Similarly, external imaging, e.g., via positron emission tomography(PET), can be used in particular to detect FAU or related compoundslabeled with ¹¹C and/or ¹⁸F. By extension from the work shown in cellculture, high levels of FAU incorporation into DNA predicts forsuccessful therapy with FAU or related compounds. Low levels of FAU inDNA suggest that an alternative therapy should be used. Thus, thepresent invention provides a method for assessing the adequacy oftreatment of tumor with various modalities, including thymidylatesynthase inhibitors, comprising administering a uridine analogue whichis labeled with a position emitter such as ¹¹C or more preferably ¹⁸Fand determining the extent of maximum TS inhibition and persistence ofTS inhibition over time between doses by external imaging preferablywith position emission tomography. These parameters can be used to guidetiming of subsequent doses or to determine when current therapy is nolonger successful and it is necessary to switch to an alternativetherapy.

In preferred embodiments for imaging applications, W may be a labelcontaining moiety or a label. The label may be any moiety that permitsthe detection of the nucleoside analogue. In preferred embodiments, thelabel includes a positron emitting atom and in a most preferredembodiment, W is ¹⁸F. In other preferred embodiments for imagingapplications, E may be H or methyl. In a most preferred embodiment forimaging, W is ¹⁸F and E is methyl or H. However, other embodiments arewithin the scope of the present invention.

It is not necessary for the nucleosides used in imaging applications tobe activated by TS. In certain embodiments, the bases of the nucleosideswill be 5-methyl deoxyuridine analogues (i.e. thymidine analogues). Byproviding the nucleoside with a methyl group at the 5-position, onebiosynthetic step required for incorporation of the nucleoside into DNAis eliminated. This may have the effect of speeding incorporation intothe target DNA and thus providing a better imaging results since thenucleosides should be incorporated more efficiently. In a preferredembodiment, the nucleoside used for imaging will be FMAU wherein thefluorine atom at the 2′-position will be ¹⁶F. Nucleosides having othermodifications at the 5-position of the base may be used in imagingapplications. For example, the 5-position of the base may be modified toinclude an iodine atom. Thus, in a preferred embodiment, the nucleosidewill be FIAU and the fluorine atom at the 2′-position will be ¹⁸F. Anyother modifications at the 5 position of the base may be used in thepractice of the imaging applications of the present invention. Inpreferred embodiments, the nucleoside can serve as a substrate for theenzymes required for incorporation of the nucleoside into DNA thus, thenucleoside will have a 5′-hydroxyl group that can be phosphorylated tothe nucleoside triphosphate and the resultant triphosphate can serve asa substrate for the cellular DNA polymerase enzymes.

Also, in the case of biopsy specimens, labeled IdUrd can be administeredand interpreted in terms of the cell culture data: high levels ofdehalogenation predicts for successful therapy with FAU or relatedcompounds; low dehalogenation suggests that alternative therapy shouldbe used. Thus, the present invention also provides a method ofdiagnosing tumors which are resistant to thymidylate synthase inhibitorsby administering IdUrd which has been labeled with either a radioisotopesuch as ¹⁴C or ³H, or more preferably, with a stable isotopic label suchas ¹³C, ²H, or ¹⁵N; preparing biopsy specimens of the tumor; anddetermining the extent of dehalogenation of IdUrd by thymidylatesynthase enzymes by examination of DNA of the tumor specimens. Basedupon these results, a therapy regimen can be suggested.

For oral administration, a dosage of between approximately 0.1 to 300mg/kg/day, and preferably between approximately 0.5 and 50 mg/kg/day isgenerally sufficient. The formulation may be presented in unit dosageform and may be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association, the active ingredient with liquid carriers orfinely divided solid carriers or both, optionally with one or moreaccessory ingredients, and then, if necessary, shaping the product.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients particularly mentioned herein, the formulations of thepresent invention may include other agents conventional in the art. Itshould also be understood that the compounds or pharmaceuticalcompositions of the present invention may also be administered bytopical, transdermal, oral, rectal or parenteral (for example,intravenous, subcutaneous or intramuscular) route or may be incorporatedinto biodegradable polymers allowing for the sustained release of thecompound, the polymers being implanted in the vicinity of the tumor orwhere the drug delivery is desired.

FIG. 1 shows the general structure of many uridine analogues. The baseconsists of uracil or various modifications. The interaction with TSoccurs at the 5-position, where the hydrogen atom is replaced by amethyl group. The endogenous substrate for TS,2′-deoxyuridine-5′-monophosphate (dUMP), is transformed to thymidinemonophosphate (dTMP). The original class of TS inhibitors,5-fluorouracil (FUra) and 5-fluorodeoxyuridine (floxuridine, FdUrd),after intracellular conversion into FdUMP, form a ternary complex withTS and block the endogenous conversion of dUMP to dTMP. Rather thanattempting to block the 5-position as with FUra and FdUrd, the presentinvention preserves the hydrogen at the 5-position, encouraging theacceptance of the methyl donation. Thus, for those deoxyuridineanalogues which are less toxic than the corresponding thymidineanalogues, TS can increase cytotoxicity. Analogues may consist ofmodifications of the base, sugar, or both. The phosphate group at the5′-position of the sugar is usually added intracellularly (e.g., viathymidine kinase), but modified phosphate groups may be pre-formed andenter the cell intact (e.g., phosphorothiates or HPMPC).

Several modifications of the bases are feasible. The hydrogen atposition 5 and the double bond connecting carbons 5 and 6 are the mostessential requirements in the base for the most preferred TS substrate.The nitrogen at position 1 is also a preferred embodiment, however itcould be replaced by a carbon, e.g., attempting a more stable linkagewith the sugar. The hydrogen attached to N3 can also be replaced withseveral functional groups, including a halogen, acyl or alkylsubstituent. The carboxyl at C2 or C4 can be replaced with a sulfur, asin 4-thiodeoxyuridine.

A phospho-sugar (or sugar analogue) must be attached to the base inorder to interact with TS. Many changes to the sugar are possible whilestill remaining a substrate for TS. In our prototypical compound, Freplaces the hydrogen atom at the 2′-position “above” the plane of thesugar (2′-F-arabino), i.e., W═F. The resulting compound, FAU, has beendemonstrated to be phosphorylated and converted to its methylated form,FMAUMP. F can also be placed below the ring at the 2′-position, X═F.Bulkier substituents at the 2′-position are synthetically possible,e.g., as reported by Verheyden J P H, Wagner D, and Moffatt J G in“Synthesis of Some Pyrimidine 2′-Amino-2′-deoxynucleosides” in J. Org.Chem. Vol 36, pages 250-254, 1971. W═OH yields uracil arabinoside, themain circulating metabolite of ara-C. Compounds substituted in the3′-position above the ring, Y, have been synthesized, e.g., as reportedby Watanabe K A, Reichman U, Chu C K, Hollenberg D H, Fox J J in“Nucleosides. 116. 1-(beta-D-Xylofuranosyl)-5-fluorocytosines with aleaving group on the 3′ position. Potential double-barreled maskedprecursors of anticancer nucleosides”in J. Med Chem 1980 Oct.;23(10):1088-1094. Below the ring at the 3′-position, Z═F produces ananalogue of fluorothymidine, an antiretroviral agent. A successfulantiviral drug, 3′-thia-cytidine (3TC) is based upon replacement of the3′-carbon with a sulfur atom, with no substituents attached above orbelow the ring. Another reported change in the sugar is the replacementof the oxygen atom with carbon, to form a carbocylic structure, e.g.,Lin T S, Zhang X H, Wang Z H, Prusoff W H, “Synthesis and AntiviralEvaluation of Carbocyclic Analogues of 2′-Azido- and2′-Amino-2′-deoxyuridine”, J. Med Chem 31:484-6, 1988.

In addition to the set of single substitutions, multiple substitutionswould also be included within the scope of the present invention. Ifboth hydrogen atoms at the 2′-position are replaced with F, theresulting molecule is 2′,2′-difluoro-deoxyuridine, which is the maincirculating metabolite from gemcitabine, 2′,2′-difluoro-deoxycytidine.

Subsequent to methylation by TS, analogues with modifications to thesugar at the 2′-position can be recognized by DNA polymerases andcompete with thymidine triphosphate (dTTP) for incorporation into DNA.If the 3′-OH is preserved (Z═OH), additional bases can be addedsubsequently. However, if Z is not —OH, then the analogue will serve asa terminator of chain growth. Both chain terminators (e.g., AZT) andnon-terminators (e.g., IdUrd) have biological activity, but the spectrumof effects can be quite different.

Acyclic sugar analogues such as acyclovir or cidofovir (HPMPC) havebiological activity. In the case of acyclovir and related molecules(such as ganciclovir), a viral form of thymidine kinase is able tophosphorylate the drug despite the altered geometry. For HPMPC, thephosphate group is already present.

Having described the invention, the following examples are given toillustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention described.It is to be clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the present invention and/or the scope of theclaims.

EXAMPLES Materials and Methods

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

Chemicals

Non-labeled and labeled FAU,; FMAU, FAUMP and dUMP were obtained fromMoravek Biochemicals, Brea, Calif. Radio labeled [2-¹⁴C]FAU;[³H-CH₃]FMAU, [5-³H]FAUMP, and [5-³H]dUMP had specific activities of0.056, 0.33, 11 and 2 Ci/mmol respectively. Deoxyribonuclease I (DNaseI) from bovine pancreas, Type II, and phosphodiesterase I from Crotalusatrox, Type VI, formaldehyde, tetrahydrofolate, 2′-deoxyuridine (dUrd),thymidine (dThd), uracil arabinoside (ara-U), and thymine arabinoside(ara-T) were obtained from Sigma Chemical Co., St. Louis, Mo. All otherreagents were analytical grade.

Cells

The human-derived cell lines CEM, MOLT-4, RAJI, U-937, K-562 and themurine-derived L1210 were purchased from the American Type CultureCollection, Rockville, Md. Cells were grown and maintained as asuspension culture in RPMI 1640 medium containing L-glutamine and 10%(v/v) heat-inactivated fetal calf serum (BRL-GIBCO, Rockville, Md.).Penicillin-streptomycin solution (Sigma Chemical Co., St. Louis, Mo.)was added to achieve a final concentration of 100 units/mL. and 100μg/mL respectively.

Methylation of FAUMP by Thymidylate Synthase in Cell Extracts

When TS adds a methyl group to the 5-position of dUMP to generate dTMP,the proton at that location is released. When [5-³H]dUMP is thesubstrate, TS activity in cell extracts can be assessed by monitoringthe accumulation rate of tritiated water. Here, [5-³H]FAUMP was used asthe substrate for methylation by TS, and the generation of FMAUMP wasdetermined from the release of tritiated water. Cell extracts wereprepared from each cell line by sonication of intact cells. (See:Armstrong R D, Diasio R B. “Improved Measurement of ThymidylateSynthetase Activity by a Modified Tritium-Release Assay” J. Biochem.Biophys. Methods. 1982; 6: 141-7 and Speth P A J, Kinsella T J, Chang AE, Klecker R W, Belanger K. Collins J M. “Incorporation ofIododeoxyuridine into DNA of Hepatic Metastases Versus Normal HumanLiver” Clin. Pharmacol. Ther. 1988; 44:369-75.) The methyl donor wasprovided by 5,10-methylene tetrahydrofolate, which was generated in situby the addition of formaldehyde to tetrahydrofolate. At various timesafter the addition of substrate (20 μM of either [5-³H]dUMP or[5-³H]FAUMP), the reaction was stopped by addition of HCl. Unreactedsubstrate was separated from tritiated water by adsorption ontoactivated charcoal. After centrifugation, an aliquot of the supernatantwas counted for tritiated water. As shown in FIG. 5, TS in cell extractsis capable of methylating FAUMP and releasing tritiated water, albeit ata slower rate than for dUMP.

Growth Inhibition Studies

All cell lines, except for L1210, were suspended in fresh media at30,000 cells/mL. L1210 cells were suspended at 10,000 cells/mL. Cells, 2mL, were added to each of the wells of 24-well plates and incubated witheither 0 to 1000 μM of FAU or 0 to 300 μM of FMAU. Incubation wasconducted at 37° C. in a humidified 5% CO₂ atmosphere for 72 hours.Inhibition of cellular growth was assessed by cell counting (Elzone 180,Particle Data, Inc., Elmhurst, Ill.). Under these conditions, thecontrol doubling times for CEM, MOLT-4, RAJI, U-937, and K-562 cellswere 21-22 hours, while the doubling times for L1210 was 8-10 hours.

Intracellular Nucleotide Formation and Incorporation into DNA

All cell lines, except for L1210, were resuspended in fresh media at300,000 cells/mL with appropriate amount of radioactive drug. L1210cells were resuspended at 150,000 cells/mL. After 24 hours at 37° C. ina humidified 5% CO₂ atmosphere, cells were harvested for nucleotidemeasurement and DNA incorporation. Soluble nucleotides were determinedfor each cell line following exposure to 10 μM FAU. Incorporation of FAUinto DNA (as FMAU) was determined over a range of FAU concentrationsfrom 1 μM to 1 mM. As described previously by Klecker, R W, Katki A G,Collins J M. in “Toxicity, Metabolism, DNA Incorporation with Lack ofRepair, and Lactate Production for1-(2′-Fluoro-2′-deoxy-β-D-arabinofuranosyl)-5-iodouracil in U-937 andMOLT-4-cells”, Mol. Pharmacol. 1994; 46;1204-1209; and Speth P A J,Kinsella T J, Chang A E, Klecker R W, Belanger K. Collins J M. in“Incorporation of Iododeoxyuridine into DNA of Hepatic Metastases VersusNormal Human Liver.” Clin. Pharmacol. Ther. 1988; 44:369-75, DNase I andphosphodiesterase I were used to release the bases from DNA. These basesand soluble nucleotides were determined by previously reportedHPLC-based methods as noted by Klecker et al. in the previouslymentioned reference, “Toxicity, Metabolism, DNA Incorporation with Lackof Repair, and Lactate Production for1-(2′-fluoro-2′-deoxy-β-D-arabinofuranosyl)-5-iodouracil in U-937 andMOLT-4-cells.” Drug incorporation into cellular DNA was determined usingthe equation: percent incorporation=100×([drug]/([dThd]+[drug]).

Dehalogenation as a Probe for Thymidylate Synthase Activity In Situ. Therelative activity of thymidylate synthase was determined afterincubation of cells with 3 μM [3H]−IdUrd for 24 hours. DNA washarvested, digested, and chromatographed as described above. Some IdUrdwas incorporated into DNA with the iodine remaining intact on thepyrimidine ring. After conversion to IdUMP, part of the IdUrd wasdehalogenated by TS to dUMP, which then was converted by TS into dTMPand subsequently incorporated into DNA, and recovered in the DNA digestas [³H−dThd]. Relative TS activity in situ was defined as the fractionof IdUrd-derived material in DNA which was dehalogenated, i.e.,([³H]−dThd)/([³H]−dThd+[³H]−IdUrd). These methods could also be usedwith non-radioactive IdUrd, if a stable isotopic label is used, e.g.,¹³C, ¹⁵N, or ²H. A mass spectrometric detector would be substituted forthe radioactivity detector in the HPLC analysis. Unlabeled IdUrd can'tbe used since its dehalogenation produced unlabeled dThd, which would beindistinguishable from the endogenous pool of dThd in DNA.

Example 1

FAUMP was converted to FMAUMP by TS in cell extracts, as demonstrated bythe accumulation of tritiated water. The rate of conversion to FMAUMPwas about 1% of the rate of dTMP formation from dUMP (FIG. 5).Continuous incubation of cells for 72 hours with the dUrd analogue, FAU,produced varying degrees of growth inhibition (FIG. 2A). At 100 μM, CEMand U-937 cells were more than 50% inhibited, MOLT-4 and K-562 weresomewhat less inhibited, but Raji and L1210 cells were completelyuninhibited.

Example 2

Continuous incubation of cells for 72 hours with the thymidine analogue,FMAU was more potent and consistently toxic. FMAU produced aconcentration-dependent inhibition of growth for all cell lines (FIG.2B). At the lowest concentration used (0.3 μM), there was a substantialeffect on the cell growth of CEM and K-562. At 100 μM all cell linesstudied were completely inhibited (>80%). The corresponding deoxyuridineanalogue, FAU, was less toxic in all cell lines and had IC₅₀ valueswhich were 10-fold higher than with FMAU (FIG. 2A). Most strikingly, thegrowth of L1210 cells, which were very sensitive to FMAU, was notinhibited by FAU, even at 1 mM.

Both FMAU and FAU were converted intracellularly into FMAU nucleotides,and subsequently incorporated into cellular DNA as FMAU(MP). Asdescribed in Table I below, CEM and U-937 cell lines were the mostefficient cell lines at forming FMAUTP from FAU and hence FMAU wasincorporated to a higher extent into the DNA of these cell lines.

TABLE I Intracellular Nucleotides Formed and Incorporation into DNA fromIncubation of Each Cell Line With 10 μM FAU for 24 Hours. nmol/10⁶ cellsDNA FAUMP FMAUTP % incorp CEM 0.96 ± 0.24 2.30 ± 0.28 0.81 ± 0.10  U-9372.70 ± 0.69 1.99 ± 0.08 0.50 ± 0.02  MOLT-4 n.d. 1.92 ± 0.55 0.19 ±0.01  K-562 11.1 ± 0.9  n.d. 0.22 ± 0.002 RAJI 0.95 ± 0.17 n.d. 0.09 ±0.002 L1210 n.d. n.d. 0.08 ± 0.005 n.d. = not detectable

This greater DNA incorporation was reflected as increased toxicity notedfor CEM and U-937 in FIG. 2A. In contrast, FAU produced lessincorporation of FMAU into the cellular DNA of L1210 cell line. This wasreflected as less than 10% decrease in growth rate, even at 1 mM. K-562cells had a noticeably higher intracellular FAUMP pool. Only traceamounts of FMAUMP or FMAUDP were found.

When cell growth was plotted versus % incorporation of FMAU in DNA (FIG.3), on the same scale used for extracellular concentration, the responsecurve was much steeper. Further, the variation among cell lines in IC50for growth inhibition in toxicity referenced to % incorporation in DNAshowed much less variation than when referenced to extracellularconcentration. Full curves were obtained for FMAU in all 6 cells lines.For FAU, due to the larger quantities of drug substance which wererequired, full curves were done in only 2 cell lines, and single pointswere obtained for the other cell lines.

Example 3

FIG. 4 presents the relative sensitivity of cell lines togrowth-inhibition by FAU compared with the activation potential for TS,measured independently as relative dehalogenation of IdUrd. The mostsensitive cell lines (U-937, CEM, MOLT-4) have 50% or moredehalogenation. The least sensitive lines (RAJI, L1210) have 15% orlower dehalogenation.

The toxicity of the compounds of the present invention has beenevaluated in an animal model system. FAU was administered by oral gavageto mice at a dose of 5 g/kg once per day for 14 consecutive days. Afteran additional 14 days of observation following this dosing period, theanimals were sacrificed. Histopathologic examination of murine tissuesfound no toxicity attributable to the drug treatment. Blood samples wereobtained at various times during this treatment, to confirm that thedrug was adequately absorbed. HPLC analysis of these samples indicatedFAU concentrations in plasma reached maximum levels of 750 micromolarand minimum levels of 50 micromolar.

Example 4

The present invention includes novel nucleoside analogues useful inimaging technologies as well as methods of synthesizing such analogues.In preferred embodiments, the nucleoside analogue will contain apositron emitting moiety. Such a moiety may be a single atom or a smallmolecule containing a positron emitting atom. In a most preferredembodiment, the positron emitting moiety will be an ¹⁸F atom.

The novel nucleoside analogues of the present invention may be preparedby a modification of the procedure reported by Tann C H, et al.,“Fluorocarbohydrates in synthesis. An efficient synthesis of1-(2-deoxy-2-fluoro-alpha-D-arabinofuranosyl)-5-iodouracil (beta-FIAU)and 1-(2-deoxy-2-fluoro-alpha-D-arabinofuranosyl) thymine (beta-FMAU).”J. Org Chem. 50:3644-47, 1985, and by the same group in U.S. Pat. No.4,879,377 issued to Brundidge, et al.

The present method of synthesizing a compound according to the presentinvention entails contacting a first molecule of the formula

wherein R₁, R₂, and R₅ may be the same or different and are blockinggroups, R₃ is a leaving group and in preferred embodiments may betriflate, mesylate, tosylate or imidazolsulfonyl, and R₄ is H, with asecond molecule containing a label under conditions causing the transferof the label to the position occupied by R4. The resulting labeledcompound is brominated at the 1 position and then condensed with amolecule of the formula

wherein

A=N, C;

B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C₁-C₆), alkoxy (C₁-C₆);

D=O, S, NH2;

E=H, or any substituent which is readily cleaved in the body to generateH.

The synthesis may be initiated with1,3,5-tri-O-benzoyl-alpha-D-ribofuranoside, which is commerciallyavailable, e.g., from Aldrich Chemical Company. This material ismodified to generate the precursor for fluorination by addition of animidazosulfonyl moiety at the 2-position in the ribo- (“down”) position.

187 mg of 1,3,5-tri-O-benzoyl-alpha-D-ribofuranoside is mixed with 1.54mL of dry methylene chloride. The mixture is protected from moisturewith a calcium chloride or calcium sulfate drying tube while cooling ina salt ice bath to −20° C. Slowly add 70 microliters (110 mg) ofsulfuryl chloride through a dropping funnel over 20 minutes. Add 0.44 mLof dry methylene chloride to wash down solids. Imidazole is added in 5equal portions totaling 10 equivalents (270 mg). Remove the reactionmixture from the cooling bath and let the reaction continue for 2 hours.As the reaction proceeds, the mixture will turn bright yellow.

After washing with water and drying with sodium sulfate, crystallizewith hexane at 0-5° C. for 16 hours. The small white crystals arecollected, dissolved in boiling acetone and filtered hot. Add boilingwater, then allow to crystallize at 4° C. for 16 hours and collect thecrystals by centrifugation.

The resultant compound has been shown to be stable for at least severalmonths at room temperature, and can be shipped to the clinical site orregional radiosynthesis center, where it can be stored until needed.

Fluorination Procedure

Because of the short half-life of ¹⁸F (110 minutes), the fluorinatednucleoside must prepared on the day of its clinical use. In thesecircumstances, the reactions steps are optimized for short times, withyield as a secondary consideration.

On the day of use, 10 mg of the imidazosulfonyl sugar is dissolved in200 microliters of acetonitrile. ¹⁸F is prepared from a cyclotron in theform of KHF₂, and 300 mCi (e.g., combined with 1.32 mg of unlabeledKHF₂) is dissolved in 50 microliters of a 1:100 dilution of acetic acid.In the presence of various organic solvents such as diethylene glycol orbutanediol, ¹⁸F from KHF₂ displaces the imidazosulfonyl moiety on thearabinose ring, and assumes the ara- (“up”) position. The preferredreaction solvent is 200 microliters of 2,3-butanediol. If a lower volumeof solvent is used (more concentrated solution of reactants), the aceticacid is not required. The preferred incubation conditions are 15 minutesat 170° C. This reaction product can be verified with authenticmaterial, available commercially in the non-radioactive form from SigmaChemical Co. A minimum of 8% yield is formed, based upon ¹⁸Fincorporation.

Although imidazosulfonyl is the preferred exchanging group forfluorination, other suitable leaving groups are equivalent for thispurpose. Examples of other, suitable groups include, but are not limitedto, triflate, mesylate and tosylate can be used instead of theimidazolsulfonyl moiety. The triflate and mesylate versions are readilyfluorinated, but the reaction of the tosylate form is less satisfactory.Those skilled in the art will appreciate that other exchanging groupsare equivalent for the purposes of the present invention. So long as theexchanging group reaction with the fluorination reagent is fast,efficient and produces minimal side products, any exchanging group knownto those in the art is equivalent. Other suitable exchanging groups aredisclosed by Berridge, et al. (1986) Int. J. Rad. Appl. Inst. Part A37(8):685-693.

Alternately, we have have shown that2-fluoro-2-deoxy-1,3,5-tri-O-benzoyl-alpha-D-arabinofuranose can beformed by direct reaction of underivatized1,3,5-tri-O-benzoyl-alpha-D-arabinofuranose with DAST,diethylamino-sulfur trifluoride, which can be produced readily with ¹⁸F.The synthesis of ¹⁸F DAST is described by Straatmann, et al. (1977) J.Nucl. Med. 18:151-158.

At the end of the reaction period, 2 mL of methylene chloride are added,followed by 2 mL of water. The methylene chloride layer is transferredto a tube containing 2 mL water. Then, the methylene chloride layer istransferred to another tube and dried under as stream of air or inertgas. Then, 400 microliters of acetonitrile, 100 microliters of aceticacid, and 30 microliters of HBr (30% w/w in acetic acid) are added. Thereaction is conducted at 125° C. for 5 minutes, producing a minimum 50%yield of 1-Br-2-F-3,5-di-O-benzoyl-alpha-D-arabinofuranose.

At the end of this reaction step, 1 mL of toluene and 0.5 mL of waterare added. The toluene layer is transferred to another tube and driedunder a stream of air or inert gas. An additional 0.5 mL toluene isadded, and dried. The bromo-fluoro-sugar is “condensed” with apyrimidine base (e.g., uracil, thymine, iodouracil) in which the 2- and4-positions have been silylated (e.g., with hexamethyldisilazane), toform bis-trimethylsilyl (TMS) derivatives. TMS-Ura is availablecommercially, from Aldrich. Other TMS-protected pyrimidine bases (e.g.,TMS-Thy, or TMS-IUra) can be prepared prior to the day of use andshipped to the site, as with the imidazosulfonyl sugar. The preparationof TMS-protected bases is described by White, et al. (1972) J. Org.Chem. 37:430. Other suitable protecting groups that can be removed afterthe reaction in conditions that do not cause a substantial deteriorationof the product may be used in place of TMS. The selection of suitableprotecting groups and the conditions for their use are well known tothose skilled in the art.

200 microliters of a solution of TMS-Ura (or other base) are dried, and1 mL of methylene chloride is added. The mixture is transferred to thetube containing the fluoro-bromo-sugar. The tube is heated at 170° C.for 15 minutes and then dried, producing a yield of at least 25% of2,4-di-TMS-3′,5′-di-O-benzoyl-2′-arabino-F-2′-deoxyuridine when TMS-Urais used.

To remove the blocking groups from the 3′- and 5′-positions of thesugar, and the 2- and 4-positions of the base, 0.3 mL of 2M ammonia inmethanol is added. The mixture is heated at 130° C. for 30 minutes. Thefinal product, e.g., ¹⁸F-FAU, is purified (e.g., using a solid-phase orliquid extraction cartridge or high-performance liquid chromatography)and prepared for administration in any pharmaceutically acceptablesolvent. Any solvent may be used that is safe when administered to asubject so long as the compound is soluble in it. Verification of theidentify of the product is obtained by comparison with authenticnonradioactive reference material using any standard technique forchemical identification. For example, FAU and FIAU are available fromMoravek Biochemicals (Brea, Calif.). FMAU also available from Moravek byspecial order (not listed in catalog).

Example 5

The labeled nucleosides of the present invention may be used to evaluatethe impact of various treatments upon tumors. Traditionally, mosttherapies (drugs and/or radiation) were directed towards decreasingtumor growth in a relatively non-specific fashion. More recently, anemphasis has been placed upon approaches such as differentiating thetumor to a slower-growing form and also preventing metastasis of thetumor. For both the traditional approach and newer approaches, a keyconsideration is early determination of the success or failure of theinitial treatment modality, with subsequent treatment modification asnecessary. Since all therapies have (substantial) side effects, thepenalty for incorrect assessment is two-fold: in addition to the loss ofvaluable time to find alternative treatment, needless toxicity isendured.

The standard tools for evaluation are often inadequate to provide timelyinformation. A tumor may actually stop growing and the active massshrink, but this success is masked by the continued presence ofnon-viable areas, such as necrotic or calcified tissue. Thus, success ofthe treatment is masked because the tumor doesn't change size byanatomically-based assessments such as X-Ray or CAT scans. Similarly,when the tumor stops responding to therapy and begins to grow, thefailure is masked initially because the viable tissue is only a minorityof the anatomically-determined lesion.

These problems can be overcome by functional imaging with the labelednucleosides of the present invention. Imaging methods using compounds ofthis type are more informative as they have the advantage of focusingonly on the viable tissue. This permits the determination of treatmentsuccess or failure even in the presence of the “noise” from nonviabletissue.

To image tumors, labeled nucleosides, preferrably labeled with ¹⁶F, areprepared as described above. The compounds can be generally administeredto a subject to be imaged at a dose of from about 1 mCi to about 60 mCi.In preferred embodiments, the labeled compounds will be administered indoses of about 1 mCi to about 20 mCi. In a most preferred embodiment,the compounds of the present inventions will be administered in doses offrom about 10 mCi to about 20 mCi. The lower limit of the dosage rangeis determined by the ability to obtain useful images. Dosages lower thanabout 1 mCi may be indicated in certain instances. The upper limit ofthe dosage range is determined by weighing the potential for radiationinduced harm to the subject against the potential value of theinformation to be gained. In certain instances, it may be necessary toadminister a dosage higher than about 60 mCi.

The radiolabeled compounds of the present invention may be administeredin any pharmaceutically acceptable solvent in which they are soluble. Inpreferred embodiments, the compounds will be dissolved in normal salineor buffered saline. The compounds of the invention may be administeredby any route known to those skilled in the art. For example, theadministration may be oral, rectal, topical, mucosal, nasal, ophthalmic,subcutaneous, intravenous, intra-arterial, parenteral, intramuscular orby any other route calculated to deliver the compound to the tissue tobe imaged. In preferred embodiments the compounds will be administeredby intravenous bolus.

Images may be acquired from about 5 minutes after administration untilabout 8 hours after administration. The maximum period in which imagesmay be acquired is determined by three factors: the physical half-lifeof ¹⁸F (110 minutes); the sensitivity of the detectors in the imagingmachinery; and the size of the dose administered. Those skilled in theart can adjust these factors to permit the acquisition of images at anappropriate time. Blood samples are also generally obtained, to confirmadequate delivery of the administered dose.

Those skilled in the art are capable of using the labeled compounds ofthe present invention to obtain useful imaging data. Details on imagingprocedures are well known and may be obtained in numerous references,for example, Lowe, et al. demonstrate the use of positron emissiontomography to analyze lung nodules (J. Clin. Oncology 16:1075-88, 1998)while Rinne, et al. demonstrate the use of an ¹⁸F-labeled probe inimaging protocols to analyze treatment efficacy in high risk melanomapatients using whole-body ¹⁸F-fluorodeoxyglucose positron emissiontomography (Cancer 82:1664-71, 1998).

Example 6

The labeled nucleosides of the present invention may be used to assessbone marrow function. Blood cells which are circulating throughout thebody have lifetimes ranging from a few days to a few months. Thus, inorder to sustain the circulation, blood cells are continuously producedby the body. The primary source of new blood cells is from bone marrow.Normally, bone marrow function can be inferred simply by examiningperipheral blood. If the number of blood cells per mL is in the normalrange and remaining steady, marrow is working satisfactorily.

In several circumstances, marrow function may have been destroyed, andit is desirable to rapidly and more directly assess the functioning ofmarrow. Since the production of new blood cells occurs via celldivision, DNA synthesis is the key step. Thus, a labeled analogue ofthymidine, such as ¹⁸F-FMAU can be used assess marrow function.

One of the circumstances in which it is desirable to rapidly obtaininformation on the status of the bone marrow is in conventionalanticancer chemotherapy. For many antitumor drugs, the bone marrow issubstantially damaged, which limits the amount of treatment which can betolerated by the patient. Thus, initially, blood cell counts droprapidly after chemotherapy, including loss of the key white blood cellswhich fight infection. Prolonged loss of these cells creates a seriousdanger of infections. Generally, the damage is repaired and blood countsreturn to normal levels within a few weeks. To overcome this delay inrecovery of circulating cells, growth factors (e.g., G-CSF, GM-CSF) areadministered to patients to stimulate more division among the cells inthe bone marrow. As reported by Sugawara, et al. (J. Clin. Oncology16(1):173-180, 1998), external imaging can provide evidence to show thatbone marrow cells are being stimulated. Sugawara, et al. used¹⁸F-fluorodeoxyglucose as a probe of general energy consumption toassess the state of the bone marrow cells. The use of a thymidineanalogue such as ¹⁸F-FMAU would be preferred because it more closelymonitors the key event, which is DNA synthesis.

In a more extreme circumstance, a patient's bone marrow is intentionallydestroyed with radiation and chemotherapy. After this treatment, thepatient receives a “bone marrow transplant”, i.e., the marrow which wasdestroyed is replaced with donor marrow from immunologically-matchedindividuals, or from a supply harvested from the patient prior totreatment. In either case, the ability of the patient to recover fromtreatment requires “engraftment”, i.e., that the injected cells enterthe marrow spaces and begin producing new cells to replace those whichhave left the circulation. There is very wide variation in the rate atwhich blood counts recover, so it is critical to determine as early aspossible if the engraftment is successful. If failure is detected early,a second bone marrow transplant can be attempted. The longer the wait todetermine if blood counts will return, the longer the period of exposureto life-threatening infections.

More recently, marrow function has been restored by transplants fromselected peripheral blood cells, known as stem cells. Regardless ofwhether the source of cells is the marrow or the peripheral circulation,engraftment can be monitored via ¹⁸F-FMAU and similar compounds.

The labeled nucleosides of the present invention can be substituted intothe procedure of Sugawara, et al. For example, 1-20 mCi of labelednucleoside may be administered to a subject. Following administration,sequential dynamic scans may be conducted for 60 minutes followinginjection. A number of scans of varying duration may be conducted. Forexample, one protocol that may be used is to conduct six 10-secondimages, three 20-second images, two 1.5-minute images, one 5-minuteimage, and five 10-minute images. Those skilled in the art willappreciate that other imaging protocols using varying number andduration of scans may used to practice the present invention.

Example 7

The labeled nucleosides of the present invention may be used to assessthe regeneration of liver after surgery. After exposure to injury(chemical, biological, physical, including surgery), the liver is one ofthe few organs of the body which has the ability to regenerate itself.Hepatocytes begin to replicate themselves to replace lost cells. Themost essential element of the regeneration process is synthesis of newDNA molecules. A labeled analogue of thymidine, such as ¹⁸F-FMAU, wouldbe ideal for tracking the rate of regeneration. The short half-life andlower energy of ¹⁸F would be advantages compared with radio-iodine basedprobes.

Unlike most therapeutic situations, in which a baseline image would becompared with changes induced by treatment, no baseline is typicallyavailable in situations in which regeneration is occurring. However, thenormal rate of DNA synthesis is very low, so regenerating tissue wouldbe readily detected. For example, Vander Borght, et al. demonstrate inrats that up to 10-fold differences in DNA synthesis can be found inregenerating liver compared with non-regenerating liver demonstratingthe feasability of this approach. Vander Borght, et al. usedradiolabeled thymidine analogues in a noninvasive measurement of liverregeneration with positron emission tomography (Gastroenterology. 1991Sep; 101(3):794-9). Further, the serial evaluation of DNA synthesis with¹⁸F-FMAU would provide excellent information regarding the stimulationor suppression of regeneration.

The procedure may consist of a bolus intravenous injection of ¹⁸F-FMAU,although other methods of administration may be used. Typically, fromabout 1 mCi to about 60 mCi of labeled compound may be administered. Inpreferred embodiments, about 10 mCi may be administered. Based upon thebiochemical processes within the liver and the physical half-life of¹⁸F, the preferred time for image acquisition would be 1-10 hours afterthe injection. Selecting the timing and duration of the scans is wellwithin the skill of the ordinary practitioner in the art.

Example 8

The labeled nucleosides of the present invention can be used to assessthe efficacy of gene therapy applications. One currently used method ofgene therapy involves introducing into a cell to be eliminated, a copyof Herpes simplex virus thymidine kinase gene (HSV-tk). The presence ofHSV-tk in the cell renders the cell sensitive to the nucleoside analogueganciclovir. In the presence of ganciclovir, cells expressing HSV-tk arekilled while those not expressing the HSV-tk gene are resistant. It haspreviously been demonstrated by Blasberg, et al. in U.S. Pat. No.5,703,056 that 2′-fluoro-arabinofuranosyl-nucleoside analogues, FIAU inparticular, are specifically phosphorylated by HSV-tk and incorporatedinto the DNA of cells expressing a functional HSV-tk. Thus, thesenucleoside analogues provide a means of assessing the incorporation ofHSV-tk into a target cell.

The radiolabeled FIAU disclosed by Blasberg, et al. has severaldisadvantages. Most notably, radioiodine is a biohazard which persistsin the body for many days. The lower energy and shorter half-life of ¹⁸Fsubstantially reduces the biohazard. In clinical situations where thegoal is to determine what is happening at the moment, the shorthalf-life of ¹⁸F-FMAU or ¹⁸F-FIAU is a clear advantage compared withradioiodine. This permits the assessment of changes that are occurringon a daily or weekly timescale.

In addition to direct application of thymidine kinase for gene therapy,thymidine kinase can also be used as a “reporter gene” to assess whetherthe vector (usually a virus) entered the target cells and becomefunctional. This is a particularly important strategy when the functionof the primary gene of interest cannot be readily assessed. In thiscase, observation of thymidine kinase with ¹⁸F-FMAU or related compoundsis a surrogate for expression of other genes.

For assessing gene therapy applications, ¹⁸F-labeled nucleosides areprepared as described above. After the gene therapy has been conductedand sufficient time to permit expression of the transduced genes, thesubject can be treated with the labeled compounds of the presentinvention. The compounds can be generally administered to a subject tobe imaged at a dose of from about 1 mCi to about 60 mCi. In preferredembodiments, the labeled compounds will be administered in doses ofabout 1 mCi to about 20 mCi. In a most preferred embodiment, thecompounds of the present inventions will be administered in doses offrom about 10 mCi to about 20 mCi. The lower limit of the dosage rangeis determined by the ability to obtain useful images. Dosages lower thanabout 1 mCi may be indicated in certain instances. The upper limit ofthe dosage range is determined by weighing the potential for radiationinduced harm to the subject against the potential value of theinformation to be gained. In certain instances, it may be necessary toadminister a dosage higher than about 60 mCi.

The radiolabeled compounds of the present invention may be administeredin any pharmaceutically acceptable solvent in which they are soluble. Inpreferred embodiments, the compounds will be dissolved in normal salineor buffered saline. The compounds of the invention may be administeredby any route known to those skilled in the art. For example, theadministration may be oral, rectal, topical, mucosal, nasal, ophthalmic,subcutaneous, intravenous, intra-arterial, parenteral, intramuscular orby any other route calculated to deliver the compound to the tissue tobe imaged. In preferred embodiments the compounds will be administeredby intravenous bolus.

Images may be acquired from about 5 minutes after administration untilabout 8 hours after administration. The maximum period in which imagesmay be acquired is determined by three factors: the physical half-lifeof ¹⁸F (110 minutes); the sensitivity of the detectors in the imagingmachinery; and the size of the dose administered. Those skilled in theart can adjust these factors to permit the acquisition of images at a anappropriate time. Blood samples may also be obtained to confirm adequatedelivery of the administered dose.

Specific examples have been set forth above to aid those skilled in theart in understanding the present invention. The specific examples areprovided for illustrative purposes only and are not to be construed aslimiting the scope of the present invention in any way. Allpublications, patents, and patent applications mentioned herein are eachincorporated by reference in their entirety, for all purposes.

What is claimed is:
 1. A method for assessing the response of tumors totreatment with a thymidylate synthase inhibitor, comprising the stepsof: (a) administering a uridine analogue selected by a method comprisingthe steps of: providing a uridine analogue unsubstituted in the5-position; testing said uridine analog for activation by thymidylatesynthase; and selecting said uridine analog when found to be activatedby thymidylate synthase; wherein said uridine analogue is labeled with apositron emitter; and (b) determining an extent of maximum thymidylatesynthase inhibition and persistence of thymidylate synthase inhibitionover time between doses by external imaging.
 2. The method of claim 1wherein the positron emitter is ¹⁸F or ¹¹C.
 3. The method of claim 1wherein the external imaging is accomplished using positron emissiontomography.
 4. The method of claim 1, wherein the uridine analoguecomprises a compound of the following general formula:

wherein: A=N, C; B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C₁-C₆),alkoxy (C₁-C₆) D=O, S, NH₂; and G=substituted or unsubstituted cyclicsugar, substituted or unsubstituted acyclic sugar, substituted orunsubstituted mono, di, or tri-phospho-cyclic-sugar phosphate;substituted or unsubstituted mono, di, or tri-phospho-acyclic-sugarphosphate; substituted or unsubstituted mono, di, or tri-phospho-cyclicsugar analogues, substituted or unsubstituted mono, di, ortri-phospho-acyclic sugar analogues wherein the substituents are alkyl(C₁-C₆), alkoxy (C₁-C₆), halogen.
 5. The method of claim 4, wherein thepositron emitter is ¹⁸F or ¹¹C.
 6. The method of claim 4, wherein theuridine analog is¹⁸F-1-(2′-fluoro-2′-deoxy-β-D-arabinofuranosyl)-uracil.
 7. The method ofclaim 1 wherein the uridine analog comprises a compound of the followingformula:

wherein: A=N, C; B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C₁-C₆),alkoxy (C₁-C₆) D=0, S, NH₂, at least one of W, X, Y, Z is a positronemitter having sufficient isotopic activity for imaging and theremainder of W, X, Y, Z=H, hydroxy, halogen, alkyl (C₁-C₆), substitutedalkyl (C₁-C₆), alkoxy (C₁-C₆), substituted alkoxy (C₁-C₆); J=C, S; andK=O, C.
 8. The method according to claim 7 wherein W is a positronemitter.
 9. The method according to claim 7 wherein W is ¹⁸F.
 10. Amethod for assessing the response of tumors to treatment with acombination of thymidylate synthase inhibitor and at least oneadditional compound with anti-tumor activity, comprising the steps of:(a) administering a combination of a uridine analogue and at least oneadditional compound with anti-tumor activity, wherein said uridineanalogue is selected by a method comprising the steps of: providing auridine analogue unsubstituted in the 5-position; testing said uridineanalog for activation by thymidylate synthase; and selecting saiduridine analog when found to be activated by thymidylate synthase;wherein said uridine analogue is labeled with a positron emitter; and(b) determining an extent of maximum thymidylate synthase inhibition andpersistence of thymidylate synthase inhibition over time between dosesby external imaging.
 11. The method of claim 10, wherein said at leastone additional compound with anti-tumor activity comprises a uridineanalog.
 12. The method of claim 1, wherein the uridine analog is amixture of enantiomers.